TSG101為腫瘤易感基因，其擁有許多生物性功能，包括參與細胞內轉錄活性之調控、調節細胞的生長分化、細胞膜的運輸及接受器的回收機制。最近報導指出，在乳突性甲狀腺癌組織及乳癌癌組織中皆發現有TSG101過量表現之現象，反之，在癌組織外圍則有TSG101表現量下降之情形，顯示人類腫瘤之形成，可能與細胞中TSG101基因表現量有關，因此了解TSG101基因表現之調控機轉，將有助於我們對細胞癌化現象之了解。為此，本實驗室已將TSG101轉錄起始點上游基因序列選殖出，並藉Luciferase重組報導質體分析，確立其近端啟動子活性區在-1~-436片段，此DNA片段經Softberry網站NSITE軟體比對分析後，我們發現序列上有MAZ、Sp1、ETF和ETS-1等轉錄因子的鍵結位，本實驗室經EMSA、定點突變及luciferase活性分析證明MAZ與Sp1為TSG101基因調控區之主要轉錄調控因子，因此，本實驗更進一步分析ETF和ETS-1等轉錄因子結合位對TSG101啟動子轉錄活性的影響。首先，我們選殖啟動子中只含有ETS-1結合序列之-190~-1 DNA片段到pGL3-basic啟動子活性分析載體 ( -190/pGL3-basic )，針對野生型及突變型-190/pGL3-basic進行luciferase活性檢測，發現ETS-1轉錄因子的鍵結位對TSG101啟動子活性扮演重要的調節角色，為了進一步確認，於是我們將啟動子區Sp1、ETF和ETS-1等轉錄因子鍵結位作不同組合之結合位定點突變，再送入COS-1、ARO、WRO等癌化的細胞株進行Luciferase啟動子報導載體之活性分析，以探討這些轉錄因子與TSG101啟動子活性的調節之相關性。結果顯示Sp1、ETF和ETS-1等轉錄因子的鍵結位對TSG101啟動子活性的調控是必須，並且彼此間有協同調控的關係，另一方面，結果也顯示Sp1及ETS-1結合位位點對TSG101啟動子活性為正向調控，ETF結合位則為負調控。

TSG101 is a tumor susceptibility gene, which exhibits many biological functions including the regulation of transcription, cell growth and differentation, protein trafficking and receptor recycling. Recent studies have revealed overexpression of TSG101 in human cancers of papillary thyroid carcinoma and breast cancer, whereas downregulation in the periphery of cancer tissue. These data indicated that the amount of TSG101 gene products might be relevant to tumor formation. Understanding the detail regulation of TSG101 gene expression might advance our knowledge on the processes of neoplastic conversion. In this regard, our lab have cloned and characterized upstream sequence of TSG101 transcription start site, and defined the region of -1~-436 as proximal promoter by luciferase reporter assay. Sequence analysis of this region using NSITE program on Softberry web site has revealed several transcription factor binding sites including MAZ, Sp1, ETF and ETS-1. Using EMSA and luciferase assay, we have demonstrated MAZ and Sp1 as major transcription factors regulate TSG101 proximal promoter. In this thesis, we advance our study to further explore the role of ETF and ETS-1 sites in regulation TSG101 promoter. We first cloned the region encompassing two ETS-1 sites in –190~-1 region into pGL3-basic( -190/pGL3-basic ). The luciferase reporter assay of wildtype and mutant –190/pGL3-basic plasmids further demonstrated important role of these ETS-1 sites. To explore detail contribution of the above mentioned transcription factor binding sites in regulation TSG101 promotor, we have made mutant reporter constructs containing different assortment of mutations in these binding sites, and assayed the effect of mutant on TSG101 promoter activity. The results showed that in cancer cell lines (COS-1, ARO and WRO) tested, Sp1, ETF, and ETS-1 binding sites are essential and they act co-operatively in regulating the activity of TSG101 proximal promoter. The results also indicated that Sp1 and ETS-1 were positive regulators, while ETF worked as a negative regulator.

中文摘要 2
英文摘要 4
背景介紹 6
實驗目的 15
實驗方法 17
結果 28
討論 34
總結及展望 43
參考文獻 44
圖表 51
附錄 60

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